JPS6013004B2 - Production method of interferon inducer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an interferon (hereinafter referred to as IF) inducer.

First, the present inventors extracted the active substance from the tissue of a plant belonging to the Compositae family, the genus Canhamus and containing an interferon-inducing active substance or its variants, and recovered the active substance from the extract. (See Japanese Patents No. 54-13024 and Akihiro-170641) to discover that it is possible to produce an IF inducer (hereinafter referred to as active substance A) having excellent IF-inducing activity and extremely low toxicity. Furthermore, it has been found that active substance A not only has an excellent inhibitory activity against tumours in humans and animals, but also has the effect of improving physiological effects (patent application filed on August 6, 1975). Plants that are used as raw materials for the production of active substance A are abundantly cultivated or grow naturally in various countries around the world, and there are varieties that naturally or artificially form variants such as mutants and hybrids.

Many plants of the genus Safflower and their variants can be used for the preparation of active substance A from many countries. The plants listed below are illustrative only. Safflower (Canhamustinc r)
Linne)
La female T female Lin ship), Canham melon arbor
eSCe ship Linne, CanhamuS side
jc female N man. Or these variables.

Since the active substance A is highly soluble in water, particularly in an alkaline aqueous solution, the active substance A is extracted from the raw material plant with water. Alternatively, it can also be extracted with a hydrophilic organic solvent. Since the molecular weight of the active substance A is about 100,000 to about 300 (mainly about 500,000 to about 100), for example, ultraviolet rays using an appropriate membrane that can separate substances with a cutoff molecular weight of 100,000 or more can be used. The active substance A can be recovered from the extract by treating the supernatant of the extract using a filtration method. Alternatively, if the extract is concentrated as desired and then a hydrophilic organic solvent is added, a precipitate containing the active ingredient is generated, from which the active substance A can be recovered. Alternatively, a suitable ammonium salt or inorganic metal salt may be used instead of the hydrophilic organic solvent. Since active substance A is a water-soluble acidic substance, the crude active substance recovered by various purification methods commonly used for the purification of this type of substance (for example, column chromatography using a gel furnace filtration agent or an ion exchange agent) Substance A
can be purified. The active substance A is contained in the largest amount in flowers among the tissues of plants of the genus Safflower or its variants, so the raw material for producing the active substance A is practically flowers. Now, as a result of subsequent research, it has been found that the seeds of these plants or their variants contain a large amount of another active substance (hereinafter referred to as active substance B) that has a superactivity equal to or higher than that of active substance A. It has been found that active substance B can be produced easily and inexpensively by a method similar to that of , and active substance A. Although the physical and chemical properties of active substance B have not yet been fully elucidated, the molecular weight of active substance A (
Active substance B has a different molecular weight (about 10,000 to about 20, mainly about 20,001 to about 8) from the seed In addition, since active substance A is mainly contained in flowers, the two are considered to be clearly different substances. Accordingly, an object of the present invention is to provide a method for producing an m-inducing agent isolated from a plant belonging to the genus Safflower or its variants.

The method for producing an IF inducer provided by the present invention is directed to
This method consists of the steps of extracting the above-mentioned active substance from the tissue of a plant belonging to the female (Female) or its mutants and containing the IF-inducing active substance, and recovering it from the extract, and is characterized by using seeds as the tissue.

According to the invention, active substances are recovered from extract fractions with molecular weights from about 10,000 to about 200,000, primarily from about 2 to about 80,000.

The method of the present invention is similar to the method for producing active substance A, but is not the same as the method for producing active substance A, since it has been modified to accommodate the unique physical and chemical properties of active substance B and seeds.

By the method of the present invention, an inducer having an IF-inducing activity equivalent to or higher than that of active substance A can be produced easily and inexpensively. Next, the method of the present invention will be explained in detail.

Raw materials Seeds of plants of the genus Safflower or their variants, which contain an IF-inducing active substance, can be used as raw materials.

Substantially, the seeds of the plants exemplified above are used, but
Particularly advantageous are safflower seeds. Safflower is a plant that is useful as a raw material, food, feed, medicinal herb, etc., and has been widely cultivated in countries around the world since ancient times. Safflower seeds are used as a raw material for edible oil in many countries. On the other hand, the most important drawback of the various known IF enhancers proposed so far is that they have high toxicity. Next, for example, safflower seeds are used industrially as a raw material for oil extraction, but the oil-based uses (for feed, etc.) of treated seeds are, so to speak, primitive. Therefore, it is easy to obtain raw materials in the quantities used in the method of the present invention at low cost and reliably. Although fresh seeds may be used as the raw material, it is advantageous to use dried seeds in terms of storage and extraction efficiency.

Seeds consist of a hard outer seed coat and its contents, both of which contain the active substance. Crushing the seeds before extraction can increase extraction efficiency.

Therefore, it is a good method to use oil cake from seeds after oil extraction as a raw material. In particular, safflower seeds contain a large amount of oil and fat, but seeds that have been processed using conventional methods for extracting safflower oil (e.g., hot press method, organic solvent extraction method, etc.), seeds that have been processed using conventional methods (for example, screw press), The oil cake of safflower seeds extracted with hexane usually contains about 1% fat and oil and is used as feed) can be used as a good raw material for the method of the present invention. When comparing the case where the active substance was defatted with an organic solvent before extracting the active substance from the seeds and the case where it was not defatted, it was found that there was no significant difference in the yield of the active substance and the IF-inducing activity. Extraction with extraction water is an inexpensive and practical method.

Extraction with water can be carried out at any temperature (e.g. from room temperature to about 130°C).
up to qo). Since the substance according to the present invention is highly soluble in alkaline aqueous solutions (e.g., Kaze 7-10), the pH of the water during extraction can be adjusted using known buffers, sodium hydroxide, potassium hydroxide, ammonium hydroxide, etc. good. Although the extraction time is arbitrary, it is usually 1-5 days at room temperature. If the extraction temperature is high, the extraction time will be shortened (for example, 40 - 120 x 0 from 30 minutes to 6 hours)
. In this way, most of the active ingredients contained in the seeds (90% or more in some cases) can be extracted. If desired, a suitable preservative can be added during extraction. Extraction may be continuous or batchwise. The ratio of extraction water to raw material is arbitrary. (For example, the volume of 5-1 of raw materials). If desired, a suitable organic solvent (e.g. methanol, ethanol,
The target product can be easily extracted by removing impurities such as lipids contained in the seeds with one or a mixture of chloroform, ether, hexane, and then extracting with water.
Moreover, the active substance is not destroyed.

The outer seed coat and its contents may be extracted separately. If desired, the active substances can also be extracted from the seeds with a mixture of phenol and water.

In this case, the concentration of phenol may be, for example, 20-60%, especially 40-50%, and the temperature and time may be arbitrary (eg, 50-80° C. for 1 minute to 60 minutes). The isobasa of the plant is removed from the extract by conventional methods such as recovery furnace filtration, squeezing, or centrifugation, and the inert components such as low molecular weight substances and pigments are removed from the resulting extract, and the active ingredients are removed. to recover.

For this purpose, an example of a practical method is as follows. Legs The molecular weight of the IF-inducing active substance according to the present invention is about 1 or more and about 20
(mainly about 2 to 8 directions), for example, the molecular weight cut-off is 10,000 to 20 directions (e.g., 20,000 to 8 directions).
The upper light is treated using an ultrafiltration method using a suitable membrane that can separate the substances. For example, the pressure in an ultra-furnace is 0.1-5k9
/ Can be used as an enemy. The active parts thus obtained are collected and lyophilized to yield a white powder.

When fractionating the liquid extracted with phenol/water by ultrafiltration, remove phenol and solvents that may damage the membrane.
It must be completely removed by appropriate techniques such as dialysis or depressurization before passing through the ultrafurnace.

Alternatively, after extraction with water and filtration in an ultrafurnace, it may be re-extracted with phenol/water. [B} Concentrate the extract under reduced pressure if desired, and add a hydrophilic organic solvent (e.g., methanol, ethanol, propanol, butanol, acetone, etc.) to the extract or its concentrate at an appropriate concentration (e.g., 40-7%). ), a precipitate containing the active ingredient is formed, which can be dialyzed, for example, in a cellulose tube, if desired, or dried after removing the organic solvent under reduced pressure to obtain a white powder.

'q Instead of the above organic solvent, ammonium chloride,
Ammonium salts such as ammonium sulfate, cetyltrimethylammonium bromide, or inorganic metal salts such as subchloride, steel chloride, at appropriate concentrations (e.g. 20-5 sails/
v%), a precipitate containing the active ingredient will be generated, so the precipitate should be dialyzed, for example, with a cetylulose tube, or filtered in an ultra-furnace through a membrane with a molecular weight cutoff of 5,000 or 10,000. After desalting and drying, a white powder is obtained.

By treating the extract as described above, most of the active ingredients in the raw materials (90% or more in some cases) can be recovered.

However, the content of impurities in the obtained dry coarse powder is the lowest in the worst method. Furthermore, the arc method is easy to operate, inexpensive, and can be carried out in a short time. Moreover, it was found that no significant side effects were observed even when the crude powder obtained by method (2) was orally administered in large quantities to animals as it was. Purification Since the active substance obtained by the method of the present invention is a water-soluble acidic substance, it can be purified by various methods commonly used for the purification of this food substance.

This crude powder is purified by conventional methods such as column chromatography using gel filtration or ion exchange agents. When a gel filtration agent is used, elution may be performed with an appropriate buffer, but usually water may be used for elution. If an ion exchange agent is used, elute with an appropriate buffer. Examples of practical gel filter agents are Sephadex G-50 to G-
up to 200, from Cefarrows wave to fat, Cefacryl S-200 or S-300 (manufactured by Pharmacia Fine Chemicals AB, Sweden), Biogel P-3
0 to P-300, Biogel A (manufactured by Biorad Laboratories, USA), Sagaback (Seraback, UK).

(manufactured by Laboratories), etc. Practical examples of ion exchangers are DEAE Cephadex A-25 and A-50.
(CI-type), QAE Cephadex A-25 and A
-50 (CI-type), CM Cephadex C-25 and C-50 (Na+ type), SP Cephadex C-25
and C-50 (Na1 type), DEAE Cefacel (C
I-type), DEAE Sepharose CL dose (CI-type)
, CM Sepharose CL-string (Na 10) (manufactured by Pharmacia Fine Chemicals AB, Sweden), and the like. The crude powder can also be purified using a suitable anion or cation exchange cellulose. Although the product thus obtained contains some impurities, it can be used as an IF inducer. If desired, impurities can be further removed by combining the above purification steps. m-induced activity IF was induced in the cells and serum of test animals using a sample of the final product obtained by the method described in the Examples below, and the activity was measured by the method described in the Test Examples below. As shown in Table and Table 2, IF hyperactivity was observed.

Table 1 IF activity (in vitro method) The results obtained from the final product of Example 1 using the method described in the test example below using 5 rabbits are shown in Table 2. Maximum activity reached.

The activity values of the final products obtained in Example 2 and below were also approximately the same. Table 2 (IF activity, in-pivot method, average value) Next, the seeds of safflower etc. used in the following examples are produced in Japan, but for comparison, the following seeds of foreign safflower were used in each case. The final products obtained by each treatment according to the methods described in the examples were treated with the in vitro method and in vivo method described in the test examples below, and the results of a stimulation test showed a significant difference from the results of Japanese safflower. was not recognized.

'11 Produced in Tucson, Arizona, U.S.A. ■ "Oila" from the University of Arizona {3} "PaniaI Hul" from the University of California (produced at the U.S. Department of Agriculture Nursery)
l'', ``14-5'', ``Red Fertilized Hiyama 1-2'' and ``01eicLee'' (4} Paris, France, commercially available product■ Indian Calcutta commercially available product described in the test examples [a','b} below) IF samples produced by in vitro and in vitro methods were tested using rabbit R of the same species.
In addition to suppressing the proliferation of varicella stomatitis virus on K-1 group cells, it also suppresses the proliferation of vaccinia virus (Vaccinia virus), but it inhibits the proliferation of varicella stomatitis virus (VSV) on L cells of mice of different animal species. Do not suppress.

Also, 0. When female trypsin is applied at 370°C for 2 hours, its IF activity is inactivated. It is therefore clear that the active substances produced by the method of the invention are substances that fall within the widely accepted definition of IF inducers. As previously mentioned, the active substance is an amorphous white powder in its substantially purified state, and is a water-soluble acidic substance. Although its physicochemical properties have not yet been fully elucidated, it is clear that the IF-induced activity is different from that of the active substance A in terms of their different molecular weights and as mentioned above.
Active substance B is a new IF.
It is believed to be an inducing agent. Active substance B is expected to be useful as a prophylactic and therapeutic agent for various viral infections in humans and animals. Example 1 Dried safflower (Care hamus tinctm;
After crushing the seeds (100 grains) of uSLin fertilizer, water (
The extract was extracted by leaving it at room temperature for 3 days in 1,000 p.m. (1,000 p.m.), centrifuged (20 min. of'
) was washed twice, and the washing liquid was combined with the extract liquid.

The extract obtained in this way was passed through an MC-4A ultrafilter (Bio Engineering K.K., Tokyo) using an XM-10
After ultrafiltration (pressure 3k9/earth) using a Yamachoshin outer furnace filtration membrane (molecular weight cutoff 10, manufactured by Amicon, USA), the permeate was subjected to PM The mixture was subjected to ultrafilter filtration using an ultrafiltration filter (manufactured by Amicon, USA). The residue was collected and lyophilized to obtain a white powder (212 o). Add this powder to water (
5'), and the aqueous solution was added to Sephadex G-1.
00 (Pharmacia Fine Chemical AB, Sweden) was added to a column (4.5 x 70) packed with water (1200 mm), and the lysate was divided into 5 sections each. A white powder (55.2 females) was obtained by desalting and lyophilizing the 6G to 15G flea.For further purification, this powder was added to 0.01M Tris-HCl buffer. (pH 7.0, 1=0.01) (5'), DEAE Sephadex A-50 (Pharmacia
Fine Chemicals AB, Sweden) was added to a column (2.5 x 70 cells) packed with 0.2M Tris-HCl buffer (Book 8.6: 500 cells containing 0.8M salt).
The eluate was divided into 5 categories, and the categories from 2 to 4 were combined. This solution was desalted in an ultrafilter using an FM-10 ultrafilter membrane (molecular weight cut off: 1, manufactured by Amicon, USA), and then lyophilized to produce an amorphous white powder (spotted.lmo). I got it. When this was compared with the first white powder, the IF mystery superactivity was increased. Example 2 Dried safflower seeds (100 seeds) were crushed and mixed with water (100 seeds).
After extracting for 60 minutes at 10 μm, the extract was treated according to the method described in Example 1 to obtain a white powder (
41.2 females) were obtained.

Example 3 Dried safflower seeds (100 seeds) were crushed and mixed with water (100 seeds).
000 M) was added and extracted in an autoclave (1 atm) at 120 oo for 60 minutes, and the mixture was treated according to the method described in Example 1 to obtain a white powder (43.4 o).

Example 4 Dried safflower seeds (100 min) were crushed and left in acetone (500 min) overnight at 4°C.
The extract was filtered through furnace paper to obtain Muzumi.

Acetone (500 ml) was added to this base liquid and extracted by shaking for 60 minutes, and the extract was filtered through furnace paper to obtain pickled vegetables. The same acetone-shaking extraction was repeated five times, and the resulting residue was dried under reduced pressure to remove acetone, water (1,000 ml) was added, and the solution was treated according to the method described in Example 1 to produce a white powder (44 ml).
．． 6 females) were obtained. Example 5 Chloroformomethanol mixture (
A white powder (30 p) was obtained by treatment in the same manner as in Example 4 except that 500'; 1:lv/v) was used.

Example 6 Safflower seeds (100 g) were squeezed with a press, and water (1000 g) was added to the residue, heated and extracted at 10 pg for 60 minutes, and treated according to the method described in Example 1. white powder (
o) of 37.6 was obtained.

Example 7 Seeds of safflower (100 grains) were crushed and mixed with water (50 grains).
0 Kite') was added and heated to a temperature of 100°C, then 68 qo of 90% phenol solution (500 Kite') was added, and while stirring,
The mixture was extracted for several minutes.

After cooling this to room temperature, it was centrifuged (700 m.p.m.
．． ; 20 minutes) was separated into an aqueous layer (upper layer), a phenol layer (middle layer, lower layer) and a residual liquid. The aqueous layer portion was collected. Another 400 parts of water was added to the mixed portion of the remaining phenol layer and Residue 11; the mixture was re-extracted at 68°C for 20 minutes. The aqueous layer at this time was collected, combined with the previous aqueous layer, placed in a cellulose tube, and dialyzed against ion-exchanged water at 4°C for 3 days. Thereafter, it was treated according to the method described in Example 1; white powder (2
50 p) were obtained. Test Example 1 IF induction method and IF activity measurement method (Reference: Y.Ko
jima, Kinesato Arch. , E×p. ,M
ed,. 43:351970) 'Ain Pitro Method for Generating M Rabbits (weighing approximately lk9, New Zealand White breed, SPF) were sacrificed by exsanguination, bone marrow and lymph node cells were collected, and calf serum was collected. Mixed cells 1 using Eagle MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10%
Make a cell suspension containing '07/' and divide each suspension section (
1') were added with 10, 1, 0.1, and 0.01 muta/' of the m-transfer agent obtained by the method described in Production Example 1 of the present invention, respectively, and cultured at 25 o'clock for 2 hours. Thereafter, each culture solution was centrifuged, and the supernatant was collected and used for measurement of activity.

'b} Aqueous solution of inducer (500%
Muta/'s [) 2M was injected into the ear vein of a rabbit (weight approximately lk9, New Zealand White breed, SPF), and 1,2,
After 4 and 6 hours, blood was collected (above 2), and the serum was used for the measurement of anti-inflammatory activity.

{Measurement of C serum activity In both the above-mentioned phantom and 'b' methods, the produced m activity is measured by the 50% black half-life method using rabbit Ken cell line (RK-13).

Claims (1)

[Scope of Claims] 1. Compositae, Safflower genus (Ca.
In the method for producing an interferon inducer, which comprises the steps of extracting the active substance from the tissue of a plant or a variant thereof belonging to P. rthamus and containing the interferon-inducing active substance, and recovering it from the extract, seeds are used as the tissue. A method characterized by using 2. The method according to claim 1, which involves extraction with water. 3. The method according to claim 1, which involves extraction with a mixture of water and phenol. 4. A method according to any one of claims 1 to 3, in which the active substance is recovered by adding a hydrophilic organic solvent to the extract. 5. A method according to any one of claims 1 to 3, in which the active substance is recovered by adding an ammonium salt or an inorganic metal salt to the extract. 6 Compositae, Safflower (Ca
rthamus) and containing the interferon-inducing active substance from the tissue of a plant or its variant, and recovering the active substance from the extract. A method for producing an interferon inducer, which comprises extracting the above-mentioned active substance with water and recovering the active substance from the extract by ultrafiltration. 7. The method according to claim 6, which comprises extracting with water containing phenol, removing the phenol from the extract, and then ultrafiltrating. 8. The method according to claim 6 or 7, in which an extract having a molecular weight of about 10,000 to about 200,000, mainly about 20,000 to about 80,000, is fractionated by ultrafiltration. 9. A method according to any one of claims 1 to 8 using pressed seeds. 10. A method according to any one of claims 1 to 9 using defatted seeds. 11. A method according to claim 10 using seeds defatted with an organic solvent. 12. The method according to claim 10, using seeds that have been defatted with an organic solvent after pressing.